There has been great commercial demand for fast, sensitive and quantitative technologies for diagnostical purposes for quite some time. These technologies are suited to be used in a very wide market area comprising of health care, research, agriculture, environmental care, veterinary care and some areas of industrial production. The increased sensitivity, high throughput, ease of use, ruggedness or lower cost per test are factors that may open new areas of use if they can be realized with these diagnostic technologies.
Certain diagnostic technologies can reach high sensitivity but at a high cost. Other methods might be commercially competitive, but they cannot be applied widely enough to serve different market areas. A technology combining the demand for high sensitivity with commercial feasibility and wide applicability will in the future have an important position and great possibilities at the diagnostic market.
At the moment diagnostic applications use several different analysis methods, e.g., radioactive labeling, enzyme linked immunoassay, colorimetric determinations, and determinations on the basis of fluorescence, chemiluminescence, and anodic or hot electron induced electrochemiluminescence (ECL). Hot electron-induced electrogenerated chemiluminescence (HECL) has been described in detail in the U.S. Pat. No. 6,251,690, Kulmala S., et al. Each of these techniques has a characteristic combination of sensitivity levels, ease of use, ruggedness, speed and operation costs, which determines the marketability. The differences in these properties come from the physical limitations of the methods. For example, the disadvantage of many applications of radioactive labeling is the weakening of the label with time as a result of radioactive fission as well as the extra costs of radioactive waste both from a safety point of view and from an environmental point of view. Application of many sensitive techniques in decentralized diagnostics is limited by the extreme complexity of the tests and the instrumentation, so that only experts can do the measurements. The complexicity of the measurement is also usually in direct relation to the cost of the instrumentation and/or the test. As an example can be mentioned, among others, anodic electrochemiluminescence (ECL), which has become a commercially popular method of detection. The instrumentations based on anodic ECL are laboratory robots with intricate features of use, so that expert knowledge is needed to use them; in addition, the measurement process includes repeated complex washing and preparation steps. All of the above mentioned things are factors increasing the cost of measurements, increasing the amount of waste and making it impossible to employ the analysis method for the practical needs of decentralized analysis.
Commercially important measurement techniques are based on the identification and the measurement of the analytes in mixtures with use of so-called labeling compounds. In measurements based on properties of biological molecules, such as immunoassays, the analyte to be measured (X) can be selectively attached from a mixture of different molecules to an antibody on a solid phase, and the attached molecules can be measured with the help of another, also compound (X) specific antibody, which has been marked, i.e., labeled with a suitable labeling compound. Examples of labeling compounds include, but are not limited to, radioactive isotopes, enzymes, light-absorbing molecules, fluorescent or phosphorescent molecules, certain metal chelates etc. The labels are attached to the antibody by means of a chemical bond. The purified compound (X) can also be labeled and used to determine the unlabeled (X) in an unknown sample by a competitive reaction. The measuring techniques of DNA and RNA are also based on selective bioaffinity and they can therefore be measured in an analogous manner. Several other chemical and biochemical analyses can be performed in this way. These days it is increasingly desirable to measure simultaneously several parameters from the sample to lower the costs and/or to increase the measurement accuracy. One possibility to achieve simultaneous measurement is to use labels that are luminescent (fluorescent or phosphorescent) at different wavelengths or have different lifetimes. Different methods and strategies of measurement, which can be used in immunodiagnostics, have been described in the book The Immunoassay Handbook, Edited by David Wild, Stockton Press Ltd., New York, 1994, pages 1-618. Naturally it is possible to measure the labeling compounds in solutions also “as is”, i.e., analyzing only the compounds when they are not used as labels.
It is previously known that organic compounds and metal chelates, which are suitable to be used as labeling compounds in analyses, can be excited with light or electrochemically so that labeling-compound specific luminescence is produced. Photoluminescence or electrochemiluminescence based techniques are usually very sensitive and well suited to excitation of labeling compounds. However, since the measured concentrations are very low, case-specific difficulties arise. The use of fluorescence can be complicated by, among others, Tyndall, Rayleigh and Raman scatterings. When measuring biological samples there appears after the excitation pulse almost without fail strong but short-lived background fluorescence. In liquid phase, phosphorescence is mainly usable only when lanthanide ions are used with specially synthesized organic molecule chelates. The problem for these techniques based on the long-lived photoluminescence of labeling compounds is the complexity and the cost of the instrumentation.
Generally the special advantages of ECL are the low cost of the electric excitation and the more simple structure (in comparison with photoluminescence), in which complex excitation optics is not needed. In addition many of the problems of photoluminescence, as described above, are avoided. The usual, so-called anodic electrochemiluminescence, in which inert metal electrodes are used, is possible to perform with organic luminophore labels with relatively simple equipment in nonaqueous solvents. However, bioaffinity assays, which are the main commercial point of interest, for the most part work only in aqueous solutions. The samples for bioaffinity assays are nearly always aqueous solutions, and thus the measuring technique for the labels has to work in water or at the very least in a micellar aqueous solution. In addition, only certain transition metal chelates can function as ECL labels in anodic ECL in aqueous or micellar aqueous solutions.
Thus far the most important commercially used anodic ECL application in analytical chemistry is a technique utilizing the derivatives of Ru(bpy)32+ chelate as labels and detecting the labels in a micellar water solution. Micellar solutions are always sensitive to various disturbing factors as a result of the uncontrolled complexity of micellar equilibria.
Hot electron-induced ECL, which is not dependent on micelles, has therefore several significant advantages in comparison to anodic ECL. Therefore the examples of this invention mainly draw upon cathodic ECL. The anodic method is also usable in both immunoassays and in DNA hybridization assays (Blackburn, G., et al., 1991, Clin. Chem. 37: 1534-1539; Kenten, J., et al. 1992, Clin. Chem. 33: 873-879). This method is currently in commercial use by Roche Diagnostics. A laboratory robot moves magnetic particles with which the label for the quantitative measurement of the analyte is transferred onto a golden continuous-use working electrode to perform immunoassays and DNA or RNA probe applications (Massey, Richard J., et al. U.S. Pat. No. 5,746,974; Leland, Jonathan K., et al. U.S. Pat. No. 5,705,402). The repeatable handling of magnetic latex particles is in many ways difficult, which is why this technique is usable only with expensive laboratory robots (e.g. Elecsys 1010 and 2010) with complex and exact liquid handling device. In addition the continuous-use massive golden working electrode demands a lengthy cleaning and pretreatment between each analysis (Elecsys Service Manual, p. 70).
Using microparticles in electroluminescence is not necessarily optimal, because as U.S. Pat. No. 5,705,402 shows, the excitation of the label substance occurs on the surface of the microparticle in contact with the gold electrode. Even though microparticles are tiny (diameter e.g. 2800 nm), only a very small part of the surface of the spherical particle can be in contact with the electrode. Consequently only a small part of the labeling substance carried by the microparticle is available for excitation. The operating efficiency is further decreased by the poor optical transparency of the magnetic material containing latex particles, since only a part of the photons can be seen at the detector at the opposite direction from the (optically non-transparent) working electrode. This direction is one where the detector is generally found in traditional analysers.
Usual anodic ECL based on traditional electrochemistry requires the use of inert metals (e.g., platinum or gold) or carbon as working electrodes. The use of these electrode materials is however limited by their narrow potential window as a result of water breaking down (oxygen produced at the anode and hydrogen at the cathode). Luminophores suitable for use as ECL labels cannot therefore be electrically excited by common means on these traditional inert electrodes, as due to the limited potential window one cannot reach high enough anodic and cathodic potentials needed for excitation reactions. This is also why anodic ECL cannot achieve the simultaneous excitation and/or time-resolved detection of several different labels, which is necessary for multiparameter assays. Anodic ECL is only suited to the needs of very large centralized laboratories. As a result of the extreme complexity and high cost of the measuring device the anodic device and technique are not suited in their previously known form to be used in the market area of decentralized analyses, e.g., during a doctors' appointment, at small health clinics, or at the patient's home.
A special disadvantage for both anodic ECL and HECL in bioaffinity assays, in addition to the problems discussed above, is the long incubation time needed to let the reactive molecules reach kinetic equilibrium, which is necessary to optimize the accuracy of the analysis. This problem can be solved fairly efficiently with the aid of porous disk devices (US 2009178924 (A1), Ala-Kleme, T. et al.). The problem with these devices is that thus far they have only included a working electrode, and the counter electrode has been inside the continuous-use cell of the electrochemiluminometer. The careful washing of the cell is imperative and sometimes very difficult, and especially the removal of anodic films forming on the counter electrode is difficult and time-consuming. As an alternative, the counter electrode would have to be placed, with difficulty, in the measuring cassette near the working electrode but so that it would not overmuch impede the light from reaching the electrode.
Hot electron electrochemistry has been utilized on flat surfaces with an insulator-covered silicon electrode or an insulator-covered aluminium electrode as the working electrode (FI 20100246, Kulmala, S., et al.) and with a conductor as the anode. The problem of hot electron electrochemistry is that there is no known electrode type that could achieve the flat plane integrated anode/cathode system with the same electrode material and reach at all as good results as with oxide-covered silicon or aluminium cathodes. The problem of silicon and aluminium is that the flat plane integrated cathode/anode chips made of these materials cannot be exploited, because labels can only be excited through a few of the first excitation pulses. After that the very fast anodic oxide layer formation soon totally prevents the current passing through the cell, and also during the formation of the oxide layer occurs a very high intensity solid state electroluminescence (also known by the old name of galvanoluminescence). Galvanoluminescence, unfortunately, also contains long-lived luminescence components, and thus time-resolved electrochemiluminescence measurement cannot be used with lanthanide chelate labels. By protecting the surface of the aluminium or silicon cathode/anode chip with an organic polymer one can diminish the problem, but it appears it is impossible to find such an electrode material that could achieve successful use of flat plane cathode/anode chips with excitation of labeling compounds in bioaffinity assays.
As we endeavoured to make a cathode/anode chip, on which the anode part of aluminium electrode glass-based chip was supposed to be covered with carbon paste to prevent anodic oxidation, by mishap both the anode and the cathode parts were covered with the carbon paste. Since the chips had been made, they were tested in measurements, and surprisingly Tb(III) chelate gave light with high amplitude electric pulses. We assumed that this was due to hot electron electrochemistry, even though tunnel emission with carbon electrodes and other conductors is not possible. For comparison we tried to excite also organic fluorophores (8-hydroxy quinolone and 7-amino-4-methyl coumarine), but none of which gave measurable ECL with the carbon electrodes. Thus it is probable that we have found a previously unknown carbon electrode related phenomenon that is suited to electric excitation or terbium chelates on a pair of carbon electrodes. Fullerenes, carbon nanotubes and similar foundations of current new carbon technologies have been found only recently, and carbon chemistry might have a lot of still unknown and unstudied parts. The excitation we described may somehow be caused by the electrode surface being in direct contact with the terbium ions, instead of the excitation occurring via the ligand. One can also speculate that the gases forming on the electrode surface (hydrogen and oxygen, especially in their atomic forms) have a significant part in the reaction mechanisms. In any case the electrochemiluminescence presented in this invention can be efficiently used in bioaffinity assays even if the reaction mechanisms are still unclear.
According to this invention one can produce electrode/electrode chips (EE chips) to replace cathode/anode chips. In EE chips it does not matter which of the two electrodes on the chip is used as the cathode and which as the anode. Production of carbon paste electrodes by printing methods is probably the cheapest possible production technique for electrodes, as a result of which this innovation is a groundbreaking one from the point of view of single-use electrochemiluminescence-based diagnostic chips and cassettes. Thus a very competitive electrochemiluminescence technique (as compared with HECL-based techniques) can be accomplished using the sort of EE chips described in this invention, which are significantly cheaper than the ones used in HECL. The methods and equipments through which the above is possible are introduced in the patent requirements 1-10.
It is also noteworthy that such a carbon electrode couple as described in this invention can be produced directly on one half of a polymer cassette, after which the said half in itself is a larger than usual EE chip. On top of this half can be integrated fluidistic operations as well as operations to take, add and filter samples as well as to add reagents. This half needs not be optically transparent. The half without the carbon electrode is by preference totally optically transparent, but it can also contain only optically transparent window part. This window formation can also, if needed, function as an optical filter if the materials are chosen accordingly.